Pulp analysis of teeth submitted to different types of forces: a histological study in rats

Abstract Objective: The purpose of this study was to histologically evaluate pulp and dentin under induced tooth movement (ITM) with different types of forces. Material and Methods: The maxillary right first molars of rats were submitted to movement with continuous (CF), continuous interrupted (CIF) and intermittent (IF) forces during 5, 7 and 9 days with nickel-titanium (NiTi) closed-coil springs exerting 50cN force magnitude. The groups were histologically evaluated as for cellularity pattern, presence of dystrophic, hemodynamic alterations in the pulp as well dentin alterations. The main observed alterations were related to hemodynamic pulp characteristics, such as presence of thrombosis, vascular congestion and hemorrhages. The hemodynamic alterations were statistically evaluated by Shapiro-Wilk normality test and analysis of variance by the Kruskall-Wallis test. Results: There was no significant differences observed between groups in the different types of applied forces and duration of ITM (vascular congestion, p=1.000; hemorrhage, p=0.305; thrombosis, p=1.000). Conclusions: Pulp tissue alterations resulting from ITM were limited to hemodynamic events, without progressing to irreversible degeneration, regardless of the type of force applied.


Introduction
Periodontal and dental pulp alterations may be induced by orthodontic movement, according to magnitude, frequency and duration of the applied force. 1 In orthodontics, the types of applied forces are continuous (CF), continuous interrupted (CIF) and intermittent (IF). 2 Studies on tissue alterations resulting from induced tooth movement (ITM), with different types of applied forces, have mainly focused on periodontal reactions and only a few have addressed pulp alterations. [3][4][5][6][7][8][9][10][11][12] The dental pulp is a highly vascularized loose connective tissue, presenting a great number of cells, extracellular matrix, blood vessels and nerve fibers. Similar to other connective tissues, the pulp has an elevated repair capacity, recovering easily under favorable conditions. The dental pulp and dentin are derived from the ectomesenchyme of the dental papilla. The coronal pulp is protected from external agents by the dentin and more externally by the enamel. On the other hand, this protection is converted into threat compromising its own existence. Thus, in face of any physical, chemical or bacterial aggressor agent, whose stimuli exceed the threshold of physiologic tolerance, an inflammatory and/or degenerative pulp response may occur. These alterations result in significant intrapulp pressure increase, due to histological characteristics of the dental pulp. 13 ITM is associated with some alterations of the dentin-pulp complex, such as rupture of the odontoblastic layer, alterations in microcirculation, hypoxia and pulp calcifications. 9,14 Depending on the type, magnitude and duration of the applied force, as well as the physiologic degree of tolerance of the dental pulp, it can be reversibly or irreversibly altered. 10 The extension of the dental pulp lesion is also correlated with the degree of the produced inflammatory response that is mediated by neuropeptides. 15 Studies have associated extrusive orthodontic forces with some harmful effects on the pulp and periodontal tissues, such as vascular stasis and pulp necrosis. 16 On the other hand, intrusive forces have been associated with vascular alterations, increase in fibrosis and calcifications. 17 In order to prevent the harmful effects on the tooth pulp, the recommended applied force magnitude in adults has been between 50 and 100 cN. 18 While some studies showed no pulp alterations resulting from orthodontic tooth movement, 19,20 other studies found several events, such as invasion of macrophages, cellular proliferation, vascular alterations and disorganization of the odontoblast layer. [3][4][5][6][7][8][9][10][11][12] A few cases of tooth vitality loss during the orthodontic treatment were reported; however, when present, they were associated with previous traumas and/or poor control of the orthodontic force. 21 Recent systematic reviews demonstrated that scientific evidences do not support the correlations between type of applied orthodontic force and dental pulp reactions. 11 Besides, only intrusive, extrusive and tipping forces were previously considered, without comparing CF, CIF and IF. 11,22 Considering the principle that biological stimuli promotes tooth movement, 23 it is important to carry out a study that examine the tissue alterations resulting from ITM, in order to better delineate the orthodontic treatment. Therefore, the purpose of this study was to histologically evaluate the dental pulp after ITM using different types of forces and treatment periods by using a suitable rat model. After that, periradicular tissues (periodontal ligament) and dental papilla were removed and the root canal was obturated with a mixture of calcium hydroxide and propylene glycol. Then, the root apex was sealed with MTA (Angelus, Londrina, PR, Brazil) and reimplantated.

Material and methods
A period of two weeks was allowed for formation of ankylosis before the second phase was initiated.
The second phase consisted of ITM of the maxillary right first molar when the rats were 76 days old, with  non-parametric test at 5% significance level (p<0.05).

Discussion
The pulp lesion observed during ITM was classified as mild and transitory, since it does not cause necrosis or abscess, but it is able to cause inflammatory reaction. 26 This process seems similar to that found in humans during orthodontic movement when intense forces are used. 5  This event, along with hypoxia, induces degenerative calcification of the dental pulp and might cause obliteration of radicular pulp. 3,8,9 However, dentin alterations such as reactional dentin deposition, dentin organization, as well as presence of dental pulp calcifications were not noticed in the present study.
Mild forces can cause a small release of CGRP, leading to an initial vascular congestion. In this study, the hemodynamic event was frequently observed in all groups; however, significant differences did not occur between the different types of forces. Nevertheless, vascular congestion can be compensated by release of angiogenic factors, preventing irreversible damages from occurring to the pulp. 15,28,29 Hemodynamic alterations such as presence of congested, thrombotic vessels and some hemorrhage areas showed an increased tendency in this study.
These events can occur due to pulp microcirculation changes, which increase tissue pressure, leading to rupture of the vessel epithelium, causing hemorrhages. 10 The hemodynamic alterations may be triggered by inflammation (irreversible pulpitis) and culminate in pulp necrosis. Coagulation necrosis may also occur due to thrombi or vessel rupture, in case of absence of blood supply. 13 Santamaria, et al. 24 (2007), evaluating pulp alterations at 6,12,24 and 72 hours after ITM, also did not observe any case of necrosis, similar to the condition observed in the present study. The effects of ITM on the pulp, already begin a few hours after the beginning of force application. 5,7 However, some alterations take a longer time to be detected, such as the deposition of reactionary dentin. Therefore, we chose to study the periods of 5, 7 and 9 days, aiming to observe not only cellular changes, but also structural alterations.
Aguiar and Arana-Chavez 30 (2007), found that after 7 days of minor trauma (extrusion), is already possible to see the start of the formation of a tubular dentine matrix (reactionary dentine). With 10 days, they observed areas of tubular reactionary dentine were coated with original odontoblasts layer. Younger dental pulps are larger, exhibiting a great number of cells with little or no fibrosis. This histological pattern changes with time and the dental pulp reduces its volume due to deposition of secondary, reparative or reactional dentin, increasing fibrosis and cellular density, besides reducing blood vessels. 13 These dynamic alterations may explain the slight pulp alteration observed in this study, given that the animals were young and the dental pulp presented a greater capacity of reaction to environmental variations, like the ITM force stress.
Furthermore, nodules and pulp calcifications are part of the natural "aging" process of the pulp, however, they can occur earlier in face of traumatic processes on the teeth structure. 31 These events were not observed in the present study, suggesting that the age of animals, intensity, type and duration of the applied forces were biologically acceptable and produced dystrophic alterations. Although the force used to promote tooth mesialization may yield tipping or bodily movement. The device used in this study promotes tipping forces, which depending on the intensity of the stimulus, can reduce blood flow, causing various damages. 32 Furthermore, studies comparing tipping and bodily forces with 10, 25, 50 e 100 g, had shown that tipping forces may cause more deleterious effects than bodily. 32,33 Orthodontic treatments with continuous force, without interruption, are frequently adopted in daily practice. Even at low force magnitude, the harmful effects may occur at higher frequency, due to either anatomical bone, dental and individual periodontal Under the conditions of the present study, it may be concluded that dental pulp alterations after ITM were limited to hemodynamic events, without significant differences between the studied groups, regardless of type and duration of applied force. No signs of progression to irreversible dental pulp degeneration were observed.